Crystal forms of azithromycin

ABSTRACT

The invention relates to novel crystal forms of azithromycin, an antibiotic useful in the treatment of infections.

BACKGROUND OF THE INVENTION

[0001] This invention relates to crystal forms of azithromycin.Azithromycin is sold commercially and is an effective antibiotic in thetreatment of a broad range of bacterial infections. The crystal forms ofthis invention are likewise useful as antibiotic agents in mammals,including man, as well as in fish and birds.

[0002] Azithromycin has the following structural formula:

[0003] Azithromycin is described and claimed in U.S. Pat. Nos. 4,517,359and 4,474,768. It is also known as9-deoxo-9a-aza-9a-methyl-9a-homoerythomycin A.

[0004] Other patents or patent applications which directly or indirectlycover azithromycin include: EP 298,650 which claims azithromycindihydrate; U.S. Pat. No. 4,963,531 which claims a method of treating astrain of Toxoplasma gondii species; U.S. Pat. No. 5,633,006 whichclaims a chewable tablet or liquid suspension pharmaceutical compositionhaving reduced bitterness; U.S. Pat. No. 5,686,587 which claims anintermediate useful in the preparation of azithromycin; U.S. Pat. No.5,605,889 which claims an oral dosage form that reduces the “foodeffect” associated with the administration of azithromycin; U.S. Pat.No. 6,068,859 which claims a controlled dosage form containingazithromycin; U.S. Pat. No. 5,498,699 which claims a compositioncontaining azithromycin in combination with bivalent or trivalentmetals; EP 925,789 which claims a method of treating eye infections;Chinese patent application CN 1123279A which relates to water solublesalts of azithromycin; Chinese patent application CN 1046945C whichrelates to azithromycin sodium dihydrogenphosphate double salts; Chinesepatent application CN 1114960A which relates to azithromycin crystals,Chinese patent application CN 1161971A which relates to azithromycincrystals; Chinese patent application CN 1205338A which relates to amethod of preparing water soluble salts of azithromycin; InternationalPublication WO 00/32203 which relates to an ethanolate of azithromycin;and European patent application EP 984,020 which relates to anazithromycin monohydrate isopropanol clathrate.

SUMMARY OF THE INVENTION

[0005] The present invention relates to crystal forms of azithromycin.As used herein, the term “crystal form(s)” or “form(s)”, unlessotherwise noted, means one or more crystal forms of azithromycin.

[0006] In particular, the present invention relates to a crystal form ofazithrocmycin wherein said crystal form is selected from forms C, D, E,F, G, H, J, M, N, 0, P, Q and R wherein said forms are as definedherein. Forms F, G, H, J, M, N, O, and P belong to family I azithromycinand belong to a monoclinic P2, space group with cell dimensions ofa=16.3±0.3 Å, b=16.2±0.3 Å, c=18.4±0.3 Å and beta=109±2°. Forms C, D, Eand R belong to family II azithromycin and belong to an orthorhombic P2₁2₁2₁ space group with cell dimensions of a=8.9±0.4 Å, b=12.3±0.5 Å andc=45.8±0.5 Å. Form Q is distinct from families I and II.

[0007] Form F azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₂H₅OHin the single crystal structure, being azithromycin monohydratehemi-ethanol solvate. Form F is further characterized as containing 2-5%water and 1-4% ethanol by weight in powder samples and having powderX-ray diffraction 2θ peaks as defined in Table 9. The ¹³C ssNMR (solidstate Nuclear Magnetic Resonance) spectrum of form F has two chemicalshift peaks at approximately 179±1 ppm, those being 179.5±0.2 ppm and178.6±0.2 ppm, a set of five peaks between 6.4 to 11.0 ppm, and ethanolpeaks at 58.0±0.5 ppm and 17.2±0.5 ppm. The solvent peaks can be broadand relatively weak in intensity.

[0008] The invention also relates to substantially pure form Fazithromycin, form F azithromycin substantially free of form Gazithromycin and form F azithromycin substantially free of azithromycindihydrate.

[0009] The invention further relates to methods of preparing form Fazithromycin by treating azithromycin with ethanol to completedissolution at 40-70° C. and cooling with reduction of ethanol oraddition of water to effect crystallization. Also included are methodsof making substantially pure form F azithromycin, form F azithromycinsubstantially free of form G azithromycin and form F azithromycinsubstantially free of azithromycin dihydrate.

[0010] Form G azithromycin is of the formula C₃₈H₇₂N₂O₁₂.1.5H₂O in thesingle crystal structure, being azithromycin sesquihydrate. Form G isfurther characterized as containing 2.5-6% water and <1% organicsolvent(s) by weight in powder samples and having powder X-raydiffraction 2θ peaks as defined in Table 9. The ¹³C ssNMR spectrum ofform G has one chemical shift peak at approximately 179±1 ppm, being apeak at 179.5±0.2 ppm (splitting <0.3 ppm may present), and a set offive peaks between 6.3 to 11.0 ppm.

[0011] The invention also relates to substantially pure form Gazithromycin, and form G azithromycin substantially free of azithromycindihydrate.

[0012] The invention further relates to methods of preparingsubstantially pure form G azithromycin, and form G azithromycinsubstantially free of azithromycin dihydrate by treating azithromycinwith a mixture of methanol and water or acetone and water to completedissolution at 40-60° C. and cooling to effect crystallization.

[0013] Form H azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.C₃H₈O₂being azithromycin monohydrate hemi-1,2 propanediol solvate.

[0014] Form J azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₃H₇OHin the single crystal structure, being azithromycin monohydratehemi-n-propanol solvate. Form J is further characterized as containing2-5% water and 1-5% 1-propanol by weight in powder samples and havingpowder X-ray diffraction 2θ peaks as defined in Table 9. The ¹³C ssNMRspectrum of form J has two chemical shift peaks at approximately 179±1ppm, those being 179.6±0.2 ppm and 178.4±0.2 ppm, a set of five peaksbetween 6.6 to 11.7 ppm and an n-propanol peak at 25.2±0.4 ppm. Thesolvent peak can be broad and relatively weak in intensity.

[0015] The invention further relates to methods of preparing form J bytreating azithromycin with n-propanol to complete dissolution at 25-55°C. and cooling with addition of water to effect crystallization.

[0016] Form M azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₃H₇OH,being azithromycin monohydrate hemi-isopropanol solvate. Form M isfurther characterized as containing 2-5% water and 1-4% 2-propanol byweight in powder samples and having powder X-ray diffraction 2θ peaks asdefined in Table 9. The ¹³C ssNMR spectrum of form M has one chemicalshift peak at approximately 179±1 ppm, being 179.6±0.2 ppm, a peak at41.9±0.2 ppm and a set of six peaks between 6.9 to 16.4 ppm and anisopropanol peak at 26.0±0.4 ppm. The solvent peak can be broad andrelatively weak in intensity.

[0017] The invention also relates to substantially pure form Mazithromycin, form M azithromycin substantially free of form Gazithromycin and form M azithromycin substantially free of azithromycindihydrate.

[0018] The invention further relates to methods of preparingsubstantially pure form M azithromycin, form M azithromycinsubstantially free of form G azithromycin and form M azithromycinsubstantially free of azithromycin dihydrate by treating azithromycinwith isopropanol to complete dissolution at 40-60° C. and reduction ofisopropanol followed by cooling or cooling followed by addition of waterto effect crystallization.

[0019] Form N azithromycin is a mixture of isomorphs of Family I. Themixture may contain variable percentages of isomorphs, F, G, H, J, M andothers, and variable amounts of water and organic solvents, such asethanol, isopropanol, n-propanol, propylene glycol, acetone,acetonitrile, butanol, pentanol, etc. The weight percent of water canrange from 1-5% and the total weight percent of organic solvents can be2-5% with each solvent content of 0.5 to 4%. The samples of form Ndisplay all characteristic peaks of members of Family I in variousproportions. Form N may be characterized as ‘mixed crystals’ or“crystalline solid solutions’ of Family I isomorphs.

[0020] Form N displays chemical shifts as a combination of isomorphs inFamily I. The peaks may vary in chemical shift ppm within ±0.2 ppm andin relative intensities and width due to the mixing of variableproportion of isomorphs contained in the form N crystalline solidsolution.

[0021] Form P azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₅H₂Obeing azithromycin monohydrate hemi-n-pentanol solvate.

[0022] Form Q azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₄H₈Obeing azithromycin monohydrate hemi-tetrahydrofuran solvate.

[0023] Form R azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.C₅H₁₂Obeing azithromycin monohydrate mono-methyl tert-butyl ether solvate.

[0024] Form D azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.C₆H₁₂ inits single crystal structure, being azithromycin monohydratemonocyclohexane solvate. Form D is further characterized as containing2-6% water and 3-12% cyclohexane by weight in powder samples and havingrepresentative powder X-ray diffraction 2θ peaks as defined in Table 9.The ¹³C ssNMR spectrum of form D displays has one chemical shift peak atapproximately 179±1 ppm, being 178.1±0.2 ppm and peaks at 103.9±0.2 ppm,95.1±0.2 ppm, 84.2±0.2 ppm, and a set of 3 peaks between 8.4 to 11 ppm.

[0025] The invention further relates to methods of preparing form D byslurrying azithromycin dihydrate with cyclohexane.

[0026] Form E azithromycin is of the formula C₃₈H₇₂N₂O₁₂.H₂O.C₄H₈O beingazithromycin monohydrate mono-tetrahydrofuran solvate.

[0027] The invention further relates to azithromycin in an amorphousstate and a method of preparing amorphous azithromycin that comprisesthe removal of water and/or solvents from the azithromycin crystallattice. The X-ray diffraction powder pattern for amorphous azithromycindisplays no sharp 20 peaks but has two broad rounded peaks. The firstpeak occurs between 40 and 13°. The second peak occurs between 130 and25°.

[0028] The invention also relates to pharmaceutical compositions for thetreatment of a bacterial infection or a protozoa infection in a mammal,fish, or bird which comprises a therapeutically effective amount of thecrystalline compounds referred to above, or amorphous azithromycin, anda pharmaceutically acceptable carrier.

[0029] The invention also relates to a method of treating a bacterialinfection or a protozoa infection in a mammal, fish, or bird whichcomprises administering to said mammal, fish or bird a therapeuticallyeffective amount of the crystalline compounds referred to above, oramorphous azithromycin.

[0030] The present invention also relates to methods of preparingcrystal forms of azithromycin which comprise the slurrying ofazithromycin in an appropriate solvent or the dissolution ofazithromycin in a heated organic solvent or organic solvent/watersolution and precipitating the crystalline azithromycin by cooling thesolution with reduction of solvent volume or by dissolving azithromycinin a solvent or solvent mixture and precipitating crystallineazithromycin by the addition of water to the solution. Azithromycin inamorphous state is prepared by heating crystalline azithromycin avacuum.

[0031] The term “treatment”, as used herein, unless otherwise indicated,means the treatment or prevention of a bacterial infection or protozoainfection as provided in the method of the present invention, includingcuring, reducing the symptoms of or slowing the progress of saidinfection. The terms “treat” and “treating” are defined in accord theforegoing term “treatment”.

[0032] The term “substantially free” when referring to a designatedcrystalline form of azithromycin means that there is less than 20% (byweight) of the designated crystalline form(s) present, more preferably,there is less than 10% (by weight) of the designated form(s) present,more preferably, there is less than 5% (by weight) of the designatedform(s) present, and most preferably, there is less than 1% (by weight)of the designated crystalline form(s) present. For instance, form Fazithromycin substantially free of azithromycin dihydrate means form Fwith 20% (by weight) or less of azithromycin dihydrate, more preferably,10% (by weight) or less of azithromycin dihydrate, most preferably, 1%(by weight) of azithromycin dihydrate.

[0033] The term “substantially pure” when referring to a designatedcrystalline form of azithromycin means that the designated crystallineform contains less than 20% (by weight) of residual components such asalternate polymorphic or isomorphic crystalline form(s) of azithromycin.It is preferred that a substantially pure form of azithromycin containless than 10% (by weight) of alternate polymorphic or isomorphiccrystalline forms of azithromycin, more preferred is less than 5% (byweight) of alternate polymorphic or isomorphic crystalline forms ofazithromycin, and most preferably less than 1% (by weight) of alternatepolymorphic or isomorphic crystalline forms of azithromycin.

[0034] The term “substantially in the absence of azithromycin dihydrate”when referring to bulk crystalline azithromycin or a compositioncontaining crystalline azithromycin means the crystalline azithromycincontains less than about 5% (by weight) azithromycin dehydrate, morepreferably less than about 3% (by weight) azithromycin dihydrate, andmost preferably less than 1% (by weight) azithromycin dihydrate.

[0035] As used herein, unless otherwise indicated, the term “bacterialinfection(s)” or “protozoa infection” includes bacterial infections andprotozoa infections and diseases caused by such infections that occur inmammals, fish and birds as well as disorders related to bacterialinfections and protozoa infections that may be treated or prevented byadministering antibiotics such as the compound of the present invention.Such bacterial infections and protozoa infections and disorders relatedto such infections include, but are not limited to, the following:pneumonia, otitis media, sinusitus, bronchitis, tonsillitis, andmastoiditis related to infection by Streptococcus pneumoniae,Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, orPeptostreptococcus spp.; pharynigitis, rheumatic fever, andglomerulonephritis related to infection by Streptcoccus pyogenes, GroupsC and G streptococci, Clostridium diptheriae, or Actinobacillushaemolyticum; respiratory tract infections related to infection byMycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae,Haemophilus influenzae, or Chlamydia pneumoniae; uncomplicated skin andsoft tissue infections, abscesses and osteomyelitis, and puerperal feverrelated to infection by Staphylococcus aureus, coagulase-positivestaphylococci (i.e., S. epidermidis, S. hemolyticus, etc.),Streptococcus pyogenes, Streptococcus agalactiae, Streptococcal groupsC-F (minute-colony streptococci), viridans streptococci, Corynebacteriumminutissimum, Clostridium spp., or Bartonella henselae; uncomplicatedacute urinary tract infections related to infection by Staphylococcussaprophyticus or Enterococcus spp.; urethritis and cervicitis; andsexually transmitted diseases related to infection by Chlamydiatrachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasmaurealyticum, or Neiserria gonorrheae; toxin diseases related toinfection by S. aureus (food poisoning and Toxic shock syndrome), orGroups A, B, and C streptococci; ulcers related to infection byHelicobacter pylori; systemic febrile syndromes related to infection byBorrelia recurrentis; Lyme disease related to infection by Borreliaburgdorferi; conjunctivitis, keratitis, and dacrocystitis related toinfection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S.pneumoniae, S. pyogenes, H. influenzae, or Listeria spp.; disseminatedMycobacterium avium complex (MAC) disease related to infection byMycobacterium avium, or Mycobacterium intracellulare; gastroenteritisrelated to infection by Campylobacter jejuni; intestinal protozoarelated to infection by Cryptosporidium spp.; odontogenic infectionrelated to infection by viridans streptococci; persistent cough relatedto infection by Bordetella pertussis; gas gangrene related to infectionby Clostridium perfringens or Bacteroides spp.; and atherosclerosisrelated to infection by Helicobacter pylori or Chiamydia pneumoniae.Also included are atherosclerosis and malaria. Bacterial infections andprotozoa infections and disorders related to such infections that may betreated or prevented in animals include, but are not limited to, thefollowing: bovine respiratory disease related to infection by P. haem.,P. multocida, Mycoplasma bovis, or Bordetella spp.; cow enteric diseaserelated to infection by E. coli or protozoa (i.e., coccidia,cryptosporidia, etc.); dairy cow mastitis related to infection by Staph.aureus, Strep. uberis, Strep. agalactiae, Strep. dysgalactiae,Klebsiella spp., Corynebacterium, or Enterococcus spp.; swinerespiratory disease related to infection by A. pleuro., P. multocida, orMycoplasma spp.; swine enteric disease related to infection by E. coli,Lawsonia intracellularis, Salmonella, or Serpulina hyodyisinteriae; cowfootrot related to infection by Fusobacterium spp.; cow metritis relatedto infection by E. coli; cow hairy warts related to infection byFusobacterium necrophorum or Bacteroides nodosus; cow pink-eye relatedto infection by Moraxella bovis; cow premature abortion related toinfection by protozoa (i.e. neosporium); urinary tract infection in dogsand cats related to infection by E. coli; skin and soft tissueinfections in dogs and cats related to infection by Staph. epidermidis,Staph. intermedius, coagulase neg. Staph. or P. multocida; and dental ormouth infections in dogs and cats related to infection by Alcaligenesspp., Bacteroides spp., Clostridium spp., Enterobacter spp.,Eubacterium, Peptostreptococcus, Porphyromonas, or Prevotella. Otherbacterial infections and protozoa infections and disorders related tosuch infections that may be treated or prevented in accord with themethod of the present invention are referred to in J. P. Sanford et al.,“The Sanford Guide To Antimicrobial Therapy,” 26th Edition,(Antimicrobial Therapy, Inc., 1996).

[0036] The present invention also includes isotopically-labeledcompounds wherein one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, and ¹⁷O. Such radiolabelled andstable-isotopically labelled compounds are useful as research ordiagnostic tools.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1 is a calculated powder X-ray diffraction pattern ofazithromycin form A. The scale of the abscissa is degrees 2-theta (2θ).The ordinate is the intensity in counts.

[0038]FIG. 2 is an experimental powder X-ray diffraction pattern ofazithromycin form A. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0039]FIG. 3 is an overlay of FIGS. 1 and 2 with the calculateddiffraction patterns of azithromycin form A (FIG. 1) on the bottom andthe experimental diffraction pattern of azithromycin form A (FIG. 2) onthe top. The scale of the abscissa is in degrees 2-theta (2θ). Theordinate is the intensity in counts.

[0040]FIG. 4 is a calculated powder X-ray diffraction pattern ofazithromycin form C. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0041]FIG. 5 is a calculated powder X-ray diffraction pattern ofazithromycin form D. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0042]FIG. 6 is an experimental powder X-ray diffraction pattern ofazithromycin form D. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0043]FIG. 7 is an overlay of FIGS. 5 and 6 with the calculateddiffraction pattern of azithromycin form D (FIG. 5) on the bottom andthe experimental diffraction pattern of azithromycin form D (FIG. 6) onthe top. The scale of the abscissa is in degrees 2-theta (2 0). Theordinate is the intensity in counts.

[0044]FIG. 8 is a calculated powder X-ray diffraction pattern ofazithromycin form E. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0045]FIG. 9 is a calculated powder X-ray diffraction pattern ofazithromycin form F. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0046]FIG. 10 is an experimental powder X-ray diffraction pattern ofazithromycin form F. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0047]FIG. 11 is an overlay of FIGS. 9 and 10 with the calculateddiffraction pattern of azithromycin form F (FIG. 9) on the bottom andthe experimental diffraction pattern of azithromycin form F (FIG. 10) onthe top. The scale of the abscissa is in degrees 2-theta (2θ). Theordinate is the intensity in counts.

[0048]FIG. 12 is a calculated powder X-ray diffraction pattern ofazithromycin form G. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity is counts.

[0049]FIG. 13 is an experimental powder X-ray diffraction pattern ofazithromycin form G. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0050]FIG. 14 is an overlay of FIGS. 12 and 13 with the calculateddiffraction pattern of azithromycin form G (FIG. 12) on the bottom andthe experimental diffraction pattern of azithromycin form G (FIG. 13) onthe top. The scale of the abscissa is in degrees 2-theta (2θ). Theordinate is the intensity in counts.

[0051]FIG. 15 is a calculated powder X-ray diffraction pattern ofazithromycin form J. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0052]FIG. 16 is an experimental powder X-ray diffraction pattern ofazithromycin form J. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0053]FIG. 17 is an overlay of FIGS. 15 and 16 with the calculateddiffraction pattern of azithromycin form J (FIG. 15) on the bottom andthe experimental diffraction pattern of azithromycin form J (FIG. 16) onthe top. The scale of the abscissa is in degrees 2-theta (2θ). Theordinate is the intensity in counts.

[0054]FIG. 18 is an experimental powder X-ray diffraction pattern ofazithromycin form M. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0055]FIG. 19 is an experimental powder X-ray diffraction pattern ofazithromycin form N. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0056]FIG. 20 is an experimental powder X-ray diffraction pattern ofamorphous azithromycin. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0057]FIG. 21 is a ¹³C solid state NMR spectrum of azithromycin form A.

[0058]FIG. 22 is a ¹³C solid state NMR spectrum of azithromycin form D.

[0059]FIG. 23 is a ¹³C solid state NMR spectrum of azithromycin form F.

[0060]FIG. 24 is a ¹³ solid state NMR spectrum of azithromycin form G.FIG. 25 is a ¹³C solid state NMR spectrum of azithromycin form J.

[0061]FIG. 26 is a ¹³C solid state NMR spectrum of azithromycin form M.

[0062]FIG. 27 is a ¹³C solid state NMR spectrum of azithromycin form N.

[0063]FIG. 28 is a ¹³C solid state NMR spectrum of amorphousazithromycin.

[0064]FIG. 29 is a ¹³C solid state NMR spectrum of a pharmaceuticaltablet containing form G azithromycin.

[0065]FIG. 30 is an experimental powder X-ray diffraction pattern ofazithromycin form Q. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0066]FIG. 31 is an experimental powder X-ray diffraction pattern ofazithromycin form R. The scale of the abscissa is in degrees 2-theta(2θ). The ordinate is the intensity in counts.

[0067]FIG. 32 is a ¹³C solid state NMR spectrum of azithromycin form H.

[0068]FIG. 33 is a ¹³C solid state NMR spectrum of azithromycin form R.

DETAILED DESCRIPTION OF THE INVENTION

[0069] Azithromycin has been found to exist in different crystallineforms. A dihydrate, form A, and a non-stroichiometric hydrate, form B,are disclosed in European Patent EP 298 650 and U.S. Pat. No. 4,512,359,respectively. Sixteen other forms have been discovered, namely forms C,D, E, F, G, H, l, J, K, L, M, N, O, P, Q and R. These forms are eitherhydrates or hydrate/solvates of azithromycin free base. Forms L and Kare the metastable lower hydrate forms of A, detected at hightemperature. Crystal structures of forms A, C, D, E, F, G, H, J and Ohave been solved. The structural data of these crystal forms are givenbelow: TABLE 1 Crystallographic data of azithromycin form A. Form AEmpirical formula C₃₈H₇₂N₂O₁₂.2H₂O Formula weight 785.2 Crystal size(mm) 0.19 × 0.24 × 0.36 Space group P2₁2₁2₁ orthorhombic Unit celldimensions a = 14.735 (5) Å b = 16.844 (7) Å c = 17.81 (1) Å α = 90° β =90° γ = 90° Z (per formula) 4 Density (g/cm³) 1.18 R 0.060

[0070] TABLE 2 Crystallographic data of azithromycin form C. Form CEmpirical formula C₃₈H₇₂N₂O₁₂.H₂O Formula weight 767.15 Crystal size(mm) 0.16 × 0.16 × 0.19 Space group P2₁2₁2₁ orthorhombic Unit celldimensions a = 8.809 (3) Å b = 12.4750 (8) Å c = 45.59 (3) Å α = 90° β =90° γ = 90° Z (per formula) 4 Density (g/cm³) 1.01 R 0.106

[0071] TABLE 3 Crystallographic data of azithromycin form D. Form DEmpirical formula C₃₈H₇₂N₂O₁₂.H₂O.C₆H₁₂ Formula weight 851.15 Crystalsize (mm) 0.52 × 0.32 × 0.16 Space group P2₁2₁2₁ orthorhombic Unit celldimensions a = 8.8710 (10) Å b = 12.506 (2) Å c = 45.697 (7) Å α = 90° β= 90° γ = 90° Z (per formula) 4 Density (g/cm³) 1.12 R 0.0663

[0072] TABLE 4 Crystallographic data of azithromycin form E. Form EEmpirical formula C₃₈H₇₂N₂O₁₂.H₂O.C₄H₈O Formula weight 839.2 Crystalsize (mm) 0.17 × 0.19 × 0.20 Space group P2₁2₁2₁ orthorhombic Unit celldimensions a = 8.869 (3) Å b = 12.086 (3) Å c = 46.00 (1) Å α = 90° β =90° γ = 90° Z (per formula) 4 Density (g/cm³) 1.13 R 0.087

[0073] TABLE 5 Crystallographic data of azithromycin form F. Form FEmpirical formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₂H₆O Crystal size (mm) 0.14 × 0.20× 0.24 Formula weight 790.2 Space group P2₁ monoclinic Unit celldimensions a = 16.281 (2) Å b = 16.293 (1) Å c = 18.490 (3) Å α = 90° β= 109.33 (1)° γ = 90° Z (per formula) 4 Density (g/cm³) 1.13 R 0.0688

[0074] TABLE 6 Crystallographic data of azithromycin form G. Form GFormula C₃₈H₇₂N₂O₁₂.1.5H₂O Formula weight 776.0 Crystal size (mm) 0.04 ×0.20 × 0.24 Space group P2₁ monoclinic Unit cell dimensions a = 16.4069(8) Å b = 16.2922 (8) Å c = 18.3830 (9) Å α = 90° β = 110.212 (2)° γ =90° Z (per formula) 4 Density (g/cm³) 1.12 R 0.0785

[0075] TABLE 7 Crystallographic data of azithromycin form H. Form HEmpirical formula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₃H₈O₂ Crystal size (mm) 0.14 ×0.20 × 0.24 Formula weight 805.0 Space group P2₁ monoclinic Unit celldimensions a = 16.177 (1) Å b = 16.241 (2) Å c = 18.614 (1) Å α = 90° β= 108.34 (1)° γ = 90° Z (per formula) 4 Density (g/cm³) 1.15 R 0.0687

[0076] TABLE 8 Crystallographic data of azithromycin form J. Form JFormula C₃₈H₇₂N₂O₁₂.H₂O.0.5C₃H₈O Formula weight 796.0 Crystal size (mm)0.40 × 0.36 × 0.20 Space group P2₁ monoclinic Unit cell dimensions a =16.191 (6) Å b = 16.237 (10) Å c = 18.595 (14) Å α = 90° β = 108.92 (4)°γ = 90° Z (per formula) 4 Density (g/cm³) 1.14 R 0.0789

[0077] TABLE 8A Crystallographic data of azithromycin form O. Form OFormula C₃₈H₇₂N₂O₁₂.0.5H₂O.0.5C₄H₁₀O Formula weight 795.04 Crystal size(mm) 0.40 × 0.36 × 0.20 Space group P2₁ monoclinic Unit cell dimensionsa = 16.3602 (11) Å b = 16.2042 (11) Å c = 18.5459 (12) Å α = 90° β =109.66 (10)° γ = 90° Z (per formula) 4 Density (g/cm³) 1.14 R 0.0421

[0078] Among these sixteen crystal forms, two isomorphic families areidentified. Family I includes forms F, G, H, J, M, N, O, and P. FamilyII includes forms C, D, E and R. Form Q is distinct from families I andII. The forms within a family are isomorphs that crystallize in the samespace group with slight variation of cell parameters and comprisechemically related structures but different elemental composition. Inthis case, the variation in chemical composition among the isomorphsarises from incorporation of different water/solvent molecules.Consequently, the isomorphs display similar but non-identical X-raydiffraction patterns and solid-state NMR spectra (ssNMR). Othertechniques such as near infrared spectroscopy (NIR), differentialscanning calorimetry (DSC), gas chromatography (GC), thermalgravimetricanalysis (TGA), or thermalgravimetric analysis/infrared spectroscopyanalysis (TG-IR), Karl Fischer water analysis (KF) and molecularmodeling/visualization provide data for affirmative identification ofisomorphs. Dehydration/desolvation temperatures were determined by DSCwith a heating rate of 5° C./min

[0079] Form C: This crystal form was identified from a single crystalstructure (Table 2)—a monohydrate of azithromycin. It has the spacegroup of P2₁2₁2₁ and similar cell parameters as that of forms D and E;therefore, it belongs to Family II isomorphs. Its calculated powderpattern is similar to that of forms D and E.

[0080] Form D: Form D was crystallized from cyclohexane. The singlecrystal structure of form D shows a stoichiometry of amonohydrate/monocyclohexane solvate of azithromycin (Table 3).Cyclohexane molecules were found to be disordered in the crystallattice. From single crystal data, the calculated water and cyclohexanecontent of form D is 2.1 and 9.9%, respectively. Both the powder patternand the calculated powder pattern of form D are similar to those offorms C and E. The powder samples of form D showed adesolvation/dehydration endotherm with an onset temperature of about 87°C. and a broad endotherm between 200-280° C. (decomposition) in DSCanalysis at 50C/min from 30-300° C.

[0081] Form D is prepared by slurrying azithromycin in cyclohexane for2-4 days. The solid form D azithromycin is collected by filtration anddried.

[0082] Form E: Form E was obtained as a single crystal collected in aTHF/water medium. It is a monohydrate and mono-THF solvate by singlecrystal analysis (Table 4). By its single crystal structure, thecalculated PXRD pattern is similar to that of form C and form D makingit a family II isomorph.

[0083] Form E is prepared by dissolving azithromycin in THF(tetrahydrofuran). Diffusing water vapor through saturated azithromycinTHF solution over time yields crystals of Form E.

[0084] Form F: The single crystal of form F crystallized in a monoclinicspace group, P2₁, with the asymmetric unit containing two azithromycin,two waters, and one ethanol, as a monohydrate/hemi-ethanolate (Table 5).It is isomorphic to all family I azithromycin crystalline forms. Thecalculated PXRD pattern of this form is similar to those of other familyI isomorphs. The theoretical water and ethanol contents are 2.3 and2.9%, respectively. The powder samples show a dehydration/desolvationendotherm at an onset temperature between 110-125° C. Form F is preparedby dissolving azithromycin in ethanol (1-3 volumes by weight) at atemperature of about 50-70° C. Upon complete dissolution, the solutionis cooled to subambient temperature to cause precipitation. The volumeof ethanol can be reduced by vacuum distillation with stirring for 1-2hours to increase the yield. Alternatively, water (optionally chilled to0-20° C.) about 0.1-2 volume can be added with collection of solidswithin 30 minute after water addition. Cooling the ethanol solution ofazithromycin prior to the addition of water to below below 20° C.,preferably below 15° C., more preferably below 10, and most preferably5° C. results in substantially pure azithromycin form F. The solid formF azithromycin is collected by filtration and dried.

[0085] Form G: The single crystal structure of form G consists of twoazithromycin molecules and three water molecules per asymmetric unit(Table 6). This corresponds to a sesquihydrate with a theoretical watercontent of 3.5%. The water content of powder samples of form G rangesfrom about 2.5 to about 6%. The total residual organic solvent is lessthan 1% of the corresponding solvent used for crystallization, which iswell below stoichiometric quantities of solvate. This form dehydrateswith an onset temperature of about 110-120° C.

[0086] Form G may be prepared by adding azithromycin to a premixedorganic solvent/water mixture (1/1 by volume), where the organic solventcan be methanol, acetone, acetonitrile, ethanol or isopropanol. Themixture is stirred and heated to an elevated temperature, e.g. 45-55° C.for 4-6 hours to cause dissolution. Precipitation occurs during coolingto ambient temperature. The solid form G azithromycin is collected byfiltration and dried.

[0087] Form H: This crystal form is a monohydrate/hemi-propylene glycolsolvate of azithromycin free base (Table 7). It was isolated from aformulation solution containing propylene glycol. The crystal structureof form H is isomorphic to crystal forms of Family I.

[0088] Azithromycin form H is prepared by dissolving azithromycindihydrate in 6 volumes of propylene glycol. To the resulting propyleneglycol solution of azithromycin, 2 volumes of water is added andprecipitation occurrs. The slurry is stirred for 24 hours and the solidsare filtered and air-dried at ambient temperature to afford crystallineForm H.

[0089] Form J: Form J is a monohydrate/hemi n-propanol solvate (Table8). The calculated solvent content is about 3.8% n-propanol and about2.3% water. The experimental data shows from about 2.5 to about 4.0%n-propanol and from about 2.5 to about 3% water content for powdersamples. Its PXRD pattern is very similar to those of its isomorphs F,G, H, M and N. Like F and G, the powder samples have adehydration/desolvation endotherm at 115-125° C.

[0090] Form J is prepared by dissolving azithromycin in 4 volumes ofn-propanol at a temperature of about 25-55° C. Water, about 6-7 volumes,is added at room temperature and the slurry is continuously stirred for0.5-2 hours. The solid form J azithromycin is collected by filtrationand dried.

[0091] Form K: The PXRD pattern of form K was found in a mixture ofazithromycin form A and microcrystalline wax after annealing at 95° C.for 3 hours. It is a lower hydrate of form A and is a metastable hightemperature form.

[0092] Form L: This form has only been observed upon heating thedihydrate; form A. In variable temperature powder X-ray diffraction(VT-PXRD) experiments, a new powder X-ray diffraction pattern appearswhen form A is heated to about 90° C. The new form, designated form L,is a lower hydrate of form A because form A loses about 2.5 weight % at90° C. by TGA, thus corresponding to a conversion to a monohydrate. Whencooled to ambient temperature, form L rapidly reverts to form A.

[0093] Form M: Isolated from an isopropanol/water slurry, form Mincorporates both water and isopropanol. Its PXRD pattern and ss-NMRspectrum are very similar to those of Family I isomorphs, indicatingthat it belongs to Family I. By analogy to the known crystal structuresof Family I isomorphs, the single crystal structure of form M would be amonohydrate/hemi-isopropranolate. The dehydration/desolvationtemperature of form M is about 115-125° C.

[0094] Form M may be prepared by dissolving azithromycin in 2-3 volumesof isopropanol (IPA) at 40-50° C. The solution is cooled to below 15°C., preferably below 10° C., more preferably about 5° C. and 2-4 volumesof cold water about 5° C. are added to effect precipitation. Seeds ofform M crystals may be added at the onset of crystallization. The slurryis stirred less than about 5 hours, preferably less than about 3 hours,more preferably less than about 1 hour and most preferably about 30minutes or less and the solids, are collected by filtration. The solidsmay be reslurried in isopropanol. This procedure provides form Msubstantially in the absence of azithromycin dihydrate.

[0095] Form N: Isolated from water/ethanol/isopropanol slurry of form A,form N crystals may contain variable amounts of the crystallizationsolvents and water. Its water content varies from about 3.4 to about 5.3weight percent. Analysis by GC Headspace reveals a variable solventcontent of ethanol and isopropanol. The total solvent content of form Nsamples is usually lower than about 5% depending on the conditions ofpreparation and drying. The PXRD pattern of form N is similar to that offorms F, G, H, J and M of the Family I isomorphs. Thedehydration/desolvation endotherm(s) of the samples of form N may bebroader and may vary between 110-130° C.

[0096] Form N azithromycin may be prepared by recrystallizingazithromycin from a mixture of azithromycin crystal latice-incorporatingorganic solvents and water, such as ethanol, isopropanol, n-propanol,acetone, acetonitirile etc. The solvent mixture is heated to 45-60° C.and azithromycin is added to the heated solvent mixture, up to a totalof about 4 volumes. Upon dissolution, 1-3 volumes of water are addedwith continuous agitation at 45-60° C. Form N azithromycin precipitatesas a white solid. The slurry is allowed to cool to ambient temperaturewith stirring. Solid form N azithromycin is isolated by filtration anddried.

[0097] Form O: This crystal form is a hemihydrate hemi-n-butanol solvateof azithromycin free base by single crystal structural data (Table 8A).It was isolated from n-butanol solution of azithromycin with diffusionof antisolvent. The crystal structure of form 0 is isomorphic to crystalforms of Family I.

[0098] Azithromycin is completely dissolved in n-butanol. Addition of anantisolvent, such as hexane, water, IPE or other non-solvent, bydiffusion results in precipitation of Form 0.

[0099] Form P: This is a proposed crystal form, being a hemihydratehemi-n-pentanol solvate of azithromycin free base. It can be isolatedfrom an n-pentanol solution of azithromycin with diffusion of anantisolvent. The crystal structure of form P is isomorphic to crystalforms of Family I.

[0100] Form P of azithromycin may be prepared as following: Azithromycinis completely dissolved in n-pentanol; addition of an antisolvent, suchas hexane, water, isopropyl ether (IPE) or other non-solvent, bydiffusion results in precipitation of Form P.

[0101] Form Q: The crystal form of Q exhibits a unique powder X-raydiffraction pattern. It contains about 4% water and about 4.5% THF,being a hydrate hemi THF solvate. The main dehydration/desolvationtemperature is from about 80 to about 110° C.

[0102] Azithromycin dihydrate is dissolved in 6 volumes of THF and 2volumes of water are added. The solution is allowed to evaporate todryness at ambient conditions to afford crystalline Form Q.

[0103] Form R: This crystalline form is prepared by adding amorphousazithromycin to 2.5 volumes of tert-butyl methyl ether (MTBE). Theresulting thick white suspension is stirred 3 days at ambientconditions. Solids are collected by vacuum filtration and air dried. Theresulting bulk azithromycin form R has a theoretical water content of2.1 weight % and a theoretical methyl tert-butyl ether content of 10.3weight %.

[0104] Due to the similarity in their structures, isomorphs havepropensity to form a mixture of the forms within a family, sometimestermed as ‘mixed crystals’ or ‘crystalline solid solution’. Form N issuch a solid crystalline solution and was found to be a mixture ofFamily I isomorphs by solvent composition and solid-state NMR data.

[0105] Both Family I and Family II isomorphs are hydrates and/orsolvates of azithromycin. The solvent molecules in the cavities havetendency to exchange between solvent and water under specificconditions. Therefore, the solvent/water content of the isomorphs mayvary to a certain extent.

[0106] The crystal forms of isomorphic Family I are more stable thanform A when subjected to heating. Forms F, G, H, J, M and N showedhigher onset dehydration temperatures at 110-125° C. than that of form Awith an onset dehydration temperature at about 90 to about 110° C. andsimultaneous solid-state conversion to form L at about 90° C.

[0107] Amorphous azithromycin: All crystal forms of azithromycin containwater or solvent(s) or both water and solvent(s). When water andsolvent(s) are removed from the crystalline solids, azithromycin becomesamorphous. Amorphous solids have advantages of high initial dissolutionrates.

[0108] The starting material for the synthesis of the various crystalforms in the examples below was azithromycin dihydrate unless otherwisenoted. Other forms of azithromycin such as amorphous azithromycin orother non-dihydrate crystalline forms of azithromycin may be used.

EXAMPLES Example Preparation of Form D

[0109] Form D was prepared by slurrying azithromycin dihydrate incyclohexane for 2-4 days at an elevated temperature, e.g. 25-50° C. Thecrystalline solids of form D were collected by filtration and dried.

Example 2 Preparation of Form F

[0110] 2A: Azithromycin dihydrate was slowly added to one volume of warmethanol, about 70° C., and stirred to complete dissolution at 65 to 70°C. The solution was allowed to cool gradually to 2-5° C. and one volumeof chilled water was added The crystalline solids were collected shortly(preferably less than 30 minutes) after addition of water by vacuumfiltration.

[0111] 2B: Azithromycin dihydrate is slowly added to one volume of warmethanol, about 70° C., and stirred to complete dissolution at 65 to 70°C. The solution is allowed to cool gradually to 2-50C and ethanol volumemay be reduced by vacuum distillation. Seeds of Form F 1-2% wt may beintroduced to facilitate the crystallization. After stirring up to 2hours the crystalline solids are collected by vacuum filtration. Theisolation of the crystals yields substantially pure form F azithromycin,form F azithromycin substantially free of form G azithromycin and form Fazithromycin substantially free of azithromycin dihydrate.

Example 3 Preparation of Form G

[0112] A reaction vessel was charged with form A azithromycin. In aseparate vessel, 1.5 volumes methanol and 1.5 volumes water were mixed.The solvent mixture was added to the reaction vessel containing the formA azithromycin. The slurry was stirred with heating to 50° C. forapproximately 5 hours. Heating was discontinued and the slurry wasallowed to cool with stirring to ambient temperature. The form Gazithromycin was collected by filtration and allowed to air dry forapproximately 30 minutes. The collected form G azithromycin was furtherdried in a vacuum oven at 45° C. This procedure yields substantiallypure form G azithromycin, and form G azithromycin substantially free ofazithromycin dihydrate.

Example 4 Preparation of Form J

[0113] Form J was prepared by dissolving azithromycin in 4 volumes ofn-propanol at a temperature of about 25° C. Water (6.7 volumes) wasadded and the slurry is continuously stirred for 1 hour, followed bycooling to about 0° C. The solid form J azithromycin was collected byfiltration and dried.

Example 5 Preparation of Form M Substantially in the Absence ofAzithromycin Dihydrate

[0114] 5A: Azithromycin dihydrate is completely dissolved in 2 volumesof warm isopropanol 40-50° C. Seeds of Form M may be optionallyintroduced to facilitate the crystallization. The solution is thencooled to 0-5° C. and 4 volumes of chilled water as antisolvent areadded and the solids are collected by vacuum filtration. The solids arereslurried in 1 volume of isopropanol for 3-5 hours at 40-45° C. andthen cooled to 0-5° C. The crystalline solids are collected shortly(about 15 minutes) after addition of water by vacuum filtration. Thesolids are reslurried in 0.5 to 1 volume of isopropanol at 25-40° C. andcooled to about 50C followed by filtration to collect solids of form M.

[0115] These procedures yield substantially pure form M azithromycin,form M azithromycin substantially free of form G azithromycin and form Mazithromycin substantially free of azithromycin dihydrate

Example 6 Preparation of Form N

[0116] Two volumes of ethanol and 2 volumes of isopropanol were added toa reaction vessel and heated to 50° C. Azithromycin form A was addedwith stirring to the heated ethanol/isopropanol mixture to yield a clearsolution. The reaction vessel was charged with 2 volumes distilled water(ambient temperature). Stirring was continued at 50° C. and solid form Nazithromycin precipitated after approximately 1 hr. Heating wasdiscontinued 5 hours after the addition of the water. The slurry wasallowed to cool to ambient temperature. Precipitated form N azithromycinwas collected by filtration and dried for 4 hours in vacuum oven at 45°C.

Example 7 Preparation of Amorphous Azithromycin

[0117] Crystalline form A azithromycin was heated to 110-120° C. in anoven for overnight under vacuum. The amorphous solids were collected andstored with desiccant as needed.

Example 8 Preparation of Form H

[0118] Azithromycin dihydrate or other crystal forms was dissolved in 6volumes of propylene glycol. To the resulting propylene glycol solutionof azithromycin, 2 volumes of water were added and precipitationoccurred. The slurry was stirred for 24 hours and the solids werefiltered and air-dried at ambient temperature to afford crystalline FormH.

Example 9 Preparation of Form Q

[0119] The crystalline powder was prepared by dissolving 500 mgazithromycin Form A in 2 ml THF. To the clear, colorless solution atroom temperature was added 1 ml water. When the solution became cloudyan additional 1 ml THF was added to dissolve the azithromycincompletely, and the solution was stirred at ambient temperature. Solventwas allowed to evaporate over 7 days, after which the dry solids werecollected and characterized.

Example 10 Powder X-Ray Diffraction Analysis

[0120] Powder patterns were collected using a Bruker D5000diffractometer (Madison, Wis.) equipped with copper radiation, fixedslits (1.0, 1.0, 0.6 mm), and a Kevex solid state detector. Data wascollected from 3.0 to 40.0 degrees in 2 theta using a step size of 0.04degrees and a step time of 1.0 seconds. The results are summarized inTable 9.

[0121] The experimental PXRD diffraction pattern of azithromycin form Ais given in FIG. 2.

[0122] The experimental PXRD diffraction pattern of azithromycin form Dis given in FIG. 6.

[0123] The experimental PXRD diffraction pattern of azithromycin form Fis given in FIG. 10.

[0124] The experimental PXRD diffraction pattern of azithromycin form Gis given in FIG. 13.

[0125] The experimental PXRD diffraction pattern of azithromycin form Jis given in FIG. 16.

[0126] The experimental PXRD diffraction pattern of azithromycin form Mis given in FIG. 18.

[0127] The experimental PXRD diffraction pattern of azithromycin form Nis given in FIG. 19.

[0128] The experimental PXRD diffraction pattern of amorphousazithromycin is given in FIG. 20.

[0129] The experimental PXRD diffraction pattern of azithromycin form Qis given in FIG. 30.

[0130] The experimental PXRD diffraction pattern of azithromycin form Ris given in FIG. 31.

[0131] The experimental variability from sample to sample is about ±0.20in 2 theta, and the same variations were observed between the calculatedpowder from single crystal structure and experimental data. Detailedanalysis showed that the isomorphs in Family I can be discerned by PXRDwith sets of characteristic peaks given in Table 9. TABLE 9 AzithromycinPowder X-ray Diffraction Peaks in 2-theta ±0.2° A D F G J M N Q  7.2 3.9  5.7  5.0  5.0  5.0  6.2  5.7  7.9  7.3  6.2  5.8  5.7  5.6  7.3 6.1  9.3  7.7  7.4  6.2  6.2  6.2  7.8  6.8  9.9 10.1  7.8  7.4  7.3 7.3  9.8  8.4 11.2 10.6  8.9  7.9  7.8  7.8 11.2  9.5 12.0 11.5  9.8 9.8  8.2  8.2 11.9 10.6 12.7 12.3 10.3 10.2  9.7  9.8 12.5 11.2 13.012.8 11.2 10.8 10.3 10.2 14.0 11.5 14.0 13.6 11.5 11.2 11.2 11.2 14.312.4 15.6 14.5 11.9 11.6 11.4 11.9 14.7 12.7 16.0 15.4 12.2 12.0 11.912.2 15.3 13.4 16.4 15.6 12.5 12.5 12.3 12.5 15.7 13.6 16.8 16.9 13.913.3 12.5 14.0 16.1 14.1 17.5 18.3 14.3 14.0 13.9 14.6 16.6 14.4 18.219.0 14.7 14.4 14.2 15.3 17.1 14.9 18.7 19.9 14.8 14.6 14.6 15.9 17.416.3 19.1 20.8 15.3 14.9 15.3 16.6 18.5 17.2 19.8 21.4 15.7 15.3 15.717.1 19.0 18.2 20.5 21.6 16.2 15.7 16.0 17.5 19.6 19.0 20.9 22.0 16.616.3 16.6 18.4 20.0 19.5 21.2 23.0 17.1 16.6 17.0 18.5 20.4 19.8 21.623.3 17.2 17.2 17.2 19.1 21.0 20.2 21.8 17.7 17.4 17.5 19.6 21.8 20.524.0 18.0 17.8 18.1 20.0 22.5 21.1 18.5 18.1 18.5 20.4 23.5 21.6 19.018.6 19.0 20.9 21.9 19.6 19.0 19.7 21.7 22.2 20.0 19.6 20.0 22.5 23.620.5 20.0 20.4 23.2 25.1 21.0 20.5 20.9 23.6 21.7 21.1 21.7 22.0 21.822.4 22.4 22.5 22.6 22.6 23.5 23.3 23.1 23.5 23.5

[0132] The peaks underlined are the characteristic peaks among forms A,D, Family I and Q.

[0133] The peaks in italic and underlined are the sets of peaks that arecharacteristic within Family I isomorphs.

[0134] Family I isomorphs have the following common characteristics: thediffraction peaks at 6.2, 11.2, 21.0±0.1 and 22.5±0.1 degree in 2-theta.Each isomorph displays representative sets of diffraction peaks given inthe following, and each set has characteristic spacing between thepeaks.

[0135] The diffraction peak positions reported are accurate to within±0.2 degree of 2-theta.

[0136] A representative PXRD pattern of form A is shown in FIG. 2. FormA displays peaks at 9.3, 13.0 and 18.7 degrees of 2-theta.

[0137] A representative PXRD pattern of form D is shown in FIG. 6. FormD displays peaks at 3.9,10.1, 10.6 and 21.4 degrees of 2-theta.

[0138] A representative PXRD pattern of Form F is shown in FIG. 10. FormF displays the characteristic peaks of Family I and three sets of peaks,being set 1 at 2-theta of 11.2 and 11.5; set 2 at 2-theta of 13.9, 14.3,14.7 and 14.8; set 3 at 2-theta of 16.2, 16.6, 17.1, 17.2 and 17.7.

[0139] A representative PXRD pattern of Form G is shown in FIG. 13. FormG displays the characteristic peaks of Family I and three sets of peaks,being set 1 at 2-theta of 11.2 and 11.6 2; set at 2-theta of 14.0, 14.4,14.6 and 14.9; set 3 at 2-theta of 16.3, 16.6, 17.2, 17.4 and 17.8.

[0140] A representative PXRD pattern of Form J is shown in FIG. 16. FormJ displays the characteristic peaks of Family I and three sets of peaks,being set 1 at 2-theta of 11.2 and 11.4; set 2 at 2-theta of 13.9, 14.2and 14.6; set 3 at 2-theta of 16.0, 16.6, 17.0, 17.2 and 17.5.

[0141] A representative PXRD pattern of Form M is shown in FIG. 18. FormM displays the characteristic peaks of Family I and three sets of peaks,being set 1 at 2-theta of 11.2; set 2 at 2-theta of 14.0 and 14.6; set 3at 2-theta of 15.9, 16.6, 17.1 and 17.5.

[0142] A representative PXRD pattern of Form N is shown in FIG. 10. FormN displays the characteristic peaks of Family I. The sets of peaks ofform N are similar to those of forms F, G, J and M, being set 1 at2-theta of 11.2 to 11.6; set 2 at 2-theta of 13.9 to 15.0; and set 3 at2-theta of 15.9 to 17.9, with the peaks may vary slightly in position,intensity and width due to mixing of variable proportion of isomorphs inFamily I.

[0143] A representative PXRD pattern of form Q is shown in FIG. 30. FormQ displays peaks at 2-theta of 6.8, 8.4 and 20.2 degree.

[0144] A representative PXRD pattern of form R is shown in FIG. 31.

Example 11 Single Crystal X-Ray Analysis

[0145] Data were collected at room temperature using Bruker X-raydiffractometers equipped with copper radiation and graphitemonochromators. Structures were solved using direct methods. The SHELXTLcomputer library provided by Bruker AXS, Inc facilitated all necessarycrystallographic computations and molecular displays (SHELXTL ReferenceManual, Version 5.1, Bruker AXS, Madison, Wis., USA (1997)).

Example 12 Calculation of PXRD Pattern from Single Crystal Data

[0146] To compare the results between a single crystal and a powdersample, a calculated powder pattern can be obtained from single crystalresults. The XFOG and XPOW computer programs provided as part of theSHELXTL computer library were used to perform this calculation.Comparing the calculated powder pattern with the experimental powderpattern confirms whether a powder sample corresponds to an assignedsingle crystal structure (Table 9A). This procedure was performed on thecrystal forms of azithromycin A, D, F, G, and J.

[0147] The calculated PXRD diffraction pattern of azithromycin form A isgiven in FIG. 1.

[0148] The calculated PXRD diffraction pattern of azithromycin form D isgiven in FIG. 5.

[0149] The calculated PXRD diffraction pattern of azithromycin form F isgiven in FIG. 9.

[0150] The calculated PXRD diffraction pattern of azithromycin form G isgiven in FIG. 12.

[0151] The calculated PXRD diffraction pattern of azithromycin form J isgiven in FIG. 15.

[0152] The results are displayed in the overlaid powder X-raydiffraction patterns for forms A, D, F, G, and J in FIGS. 3, 7, 11, 14and 17, respectively. The lower pattern corresponds to the calculatedpowder pattern (from single crystal results) and the upper patterncorresponds to a representative experimental powder pattern. A matchbetween the two patterns indicated the agreement between powder sampleand the corresponding single crystal structure.

[0153] Table 9A: Cacluated and Experimental PXRD Peaks of lsomorphs ofFamily I F calculated F experimental G calculated G experimental Jcalculated J experimental M experimental 5.2 5.0 5.7 5.8 5.8 5.7 5.6 6.36.2 6.2 6.2 6.3 6.2 6.2 7.4 7.4 7.5 7.4 7.4 7.3 7.3 7.9 7.8 7.9 7.9 7.97.8 7.8 8.8 8.9 8.9 9.3 8.3 8.2 8.2 9.9 9.8 9.9 9.9 9.8 9.7 9.8 10.310.3 10.2 10.4 10.3 10.2 10.9 10.9 10.8 11.3 11.2 11.3 11.2 11.2 11.211.2 11.5 11.4 11.6 11.6 11.4 11.4 missing 12.0 11.9 12.0 11.9 12.0 11.911.9 12.3 12.2 12.3 12.3 12.3 12.2 12.6 12.5 12.5 12.5 12.6 12.5 12.514.0 14.0 13.4 13.3 14.0 13.9 14.0 14.3 14.3 14.1 14.0 14.2 14.2 missing14.4 14.4 14.7 14.7 14.7 14.6 14.7 14.6 14.6 14.9 14.8 14.9 14.9 14.815.4 15.3 15.4 15.3 15.3 15.3 15.3 15.8 15.7 15.7 15.7 15.8 15.7 15.916.2 16.2 16.3 16.3 16.0 16.0 missing 16.6 16.6 16.6 16.6 16.7 16.6 16.617.1 17.2 17.1 17.1 17.0 17.1 17.3 17.3 17.3 17.2 17.4 17.2 missing 17.517.4 17.5 17.4 17.6 17.5 17.5 17.7 17.7 17.9 17.8 17.9 18.0 18.0 18.118.1 18.2 18.1 18.4 18.6 18.5 18.7 18.7 18.5 18.5 18.5 19.1 19.0 19.119.0 19.1 19.0 19.1 19.7 19.6 19.6 19.6 19.8 19.7 19.6 20.0 20.0 20.020.0 20.1 20.0 20.0 20.5 20.4 20.6 20.5 20.5 20.4 20.4 21.1 21.0 21.221.0 20.8 20.9 20.9 21.8 21.7 21.6 21.6 21.7 21.7 22.1 22.0 21.8 21.821.8 22.5 22.4 22.3 22.2 22.5 22.4 22.5 22.7 22.6 22.5 22.5 22.8 22.623.1 23.1 22.9 23.4 23.3 23.2 23.6 23.5 23.5 23.5 23.7 23.5 23.6

Example 13 Solid State NMR Analysis

[0154] Solid State NMR analysis:

[0155] All ¹³C solid state NMR spectra were collected on an 11.75 Tspectrometer (Bruker Biospin, Inc., Billerica, Mass.), corresponding to125 MHz ¹³C frequency. The spectra were collected using across-polarization magic angle spinning (CPMAS) probe operating atambient temperature and pressure. Depending on the quantity of sampleanalyzed, 7 mm BL or 4 mm BL Bruker probes were employed, accomodating300 mg and 75 mg of sample with maximum speeds of 7 kHz and 15 kHz,respectively. Data were processed with an exponential line broadeningfunction of 5.0 Hz. Proton decoupling of 65 kHz and 100 kHz were usedwith the 7 mm and 4 mm probes, respectively. A sufficient number ofacquisitions were averaged out to obtain adequate signal-to-noise ratiosfor all peaks. Typically, 600 scans were acquired with recycle delay of3.0 s (seconds), corresponding approximately to a 30 minute totalacquisition time. Magic angle was adjusted using KBr powder according tostandard NMR vendor practices. The spectra were referenced relative toeither the methyl resonace of hexamethylbenzen (HMB) at 17.3 ppm or theupfield resonance of adamantane (ADM) at 29.5 ppm. HMB referencedspectra show chemical shifts of all peaks shifted down field by 0.08 ppmwith respect to same spectra referenced to ADM. The spectral windowminimally included the spectra region from 190 to 0 ppm. The results aresummarized in table 10. Ss-NMR spectra for forms M, H and R werereferenced to ADM. Ss-NMR spectra for forms A, D, G, F, J and N werereferenced to HMB. Forms H and R were spun at a rate of 15 kHz. TABLE 10¹³C ss-NMR chemical shifts of Azithromycin (±0.2 ppm) A D G F J M N H R178.1  178.1  179.5* 179.5  179.6  179.6  179.6  179.5  177.9  104.1 103.9  105.5  178.6  178.4  105.6  178.7  178.7  104.6  98.4 95.1 103.5 105.5  105.5  103.4  105.6  105.4  103.6  84.6 84.2 95.0 103.4  103.4 94.9 103.6  103.2  95.3 82.6 79.4 86.2 94.9 95.0 86.7 95.0 95.0 85.479.3 78.9 83.1 86.4 86.4 82.9 86.5 86.4 84.0 78.3 75.7 78.9 83.0 82.979.3 83.1 82.7 79.4 75.6 74.6 78.2 79.1 79.2 78.1 79.0 79.2 79.0 74.774.0 77.6 78.1 78.1 77.0 77.9 78.3 75.6 73.9 72.9 76.4 77.9 76.8 76.776.5 78.0 74.5 73.5 71.9 75.7 76.5 76.2 74.7 74.8 76.4 73.9 70.8 71.074.7 74.7 74.7 74.2 74.2 74.7 73.9 68.0 69.4 74.3 74.1  74.1** 71.3 73.674.1 72.9 66.2 67.8 73.5 73.5 72.0 69.2 71.5 73.5 71.8 63.8 65.7 71.371.4 71.3 68.6 69.2 73.1 71.0 63.2 64.7 69.1 69.1 69.2 67.3 68.7 71.269.1 52.2 49.2 68.8 68.6 68.6 66.2 67.3 69.1 67.5 44.3 45.8 67.4 67.3 67.3** 65.5 66.2 68.4 65.6 42.6 43.1 65.9 66.1  66.2** 63.8 65.7 67.364.5 41.7 40.6 65.2 65.6  65.5** 63.3 63.7 66.9 49.4 39.1 37.1 64.0 63.663.7 50.0 58.1 66.1 45.7 35.4 36.4 63.3 58.0 50.0 47.1 50.1  65.5* 42.934.6 29.6 50.0 50.0 46.9 45.9 47.1  63.7* 41.6 26.9 29.3 46.9 47.0 45.944.7 46.0 49.9 40.4 26.3 28.0 46.0 45.9 44.7 43.8 44.8 46.8 37.0 23.727.7 44.5 44.7 43.7 41.9 43.8 45.9 36.2 23.3 22.1 43.7 43.7 41.6 41.141.5 44.5 29.4 21.7 21.1 41.5 41.5 41.0 37.4 41.1  43.8* 29.0 19.5 18.640.8 41.1 37.1 36.2 37.3 41.7 28.2 17.5 16.7 37.5 37.3  36.5** 33.6 36.540.9 27.4 15.9 16.1 36.5 36.4  35.4** 30.1 33.7 37.1 21.4 13.2 10.6 33.633.6 33.5 28.1 30.4 36.3 20.8 11.3  9.0 30.0 30.3 30.4 27.2 28.1 33.718.7  7.2  8.6 27.9 28.0 28.0 26.0 27.2 33.3 16.5 27.3 27.1 27.1 23.226.0  30.5* 16.1 23.1 23.2 25.2 22.8 23.2 27.9 15.7 22.5 22.6 23.2 22.522.6 27.1 10.3 21.9 21.9  22.5** 21.8 22.0 23.1  9.6

[0156] The chemical shifts labeled in bold and underlined are the peaksor sets of peaks representative of each form. The chemical shiftslabeled in italic are the solvent peaks that may be broad and variable(±0.4 ppm). The chemical shifts labeled with single asterisk may showsplitting of <0.3 ppm. The chemical shifts labeled with double asterisksmay show variation of ±0.3 ppm TABLE 10 (continued). ¹³C ss-NMR chemicalshifts of Azithromycin (±0.2 ppm) A D G F J M N H R 20.9 20.8  21.9**20.2 20.8 22.6 8.9 20.2 20.4 20.7 18.9 19.0 22.3 8.6 18.8 18.9 18.9 17.416.9 21.9 17.0 16.8 16.8 16.3 15.8 20.7 16.0 17.2  15.6** 15.5 12.2 20.312.2 15.7 12.1 12.1  9.9 18.8 10.4 12.2 11.5 10.3  9.4 17.1  9.9 10.112.1  9.6  7.9 16.6  9.3  9.8 10.0  9.3  6.6 15.8  7.6  9.3  9.3  7.715.4  6.5  7.9  8.1  7.1 12.0  6.6   6.8**  9.9  9.1  7.9  7.0

[0157] The chemical shifts labeled in bold and underlined are the peaksor sets of peaks representative of each form. The chemical shiftslabeled in italic are the solvent peaks that may be broad and variable(±0.4 ppm). The chemical shifts labeled with single asterisk may showsplitting of <0.3 ppm. The chemical shifts labeled with double asterisksmay show variation of ±0.3 ppm

[0158] The chemical shifts reported are accurate to within ±0.2 ppmunless otherwise indicated.

[0159] A representative ¹³C ssNMR spectrum of form A is shown in FIG.21. Form A displays a peak at 178.1 ppm, and peaks at 104.1, 98.4, 84.6,26.9, 13.2, 11.3 and 7.2 ppm.

[0160] A representative ¹³C ssNMR spectrum of form D is shown in FIG.22. Form D displays the highest chemical shift peak of 178.1 ppm andpeaks at chemical shifts of 103.9, 95.1, 84.2, 10.6, 9.0 and 8.6 ppm.

[0161] A representative ¹³C ssNMR spectrum of form F is shown in FIG.23. Form F has two chemical shift peaks at approximately 179.1±2 ppm,being 179.5 ppm and 178.6 ppm, and a set of 5 peaks at 10.1, 9.8, 9.3,7.9, and 6.6 ppm, and ethanol peaks at 58.0±0.5 ppm and 17.2±0.5 ppm.The solvent peaks can be broad and relatively weak in intensity.

[0162] A representative ¹³C ssNMR spectrum of form G is shown in FIG.24. Form G has the highest chemical shift peak of 179.5 ppm, being asingle peak with possible splitting of <0.3 ppm and a set of 5 peaks at10.4, 9.9, 9.3, 7.6, 6.5 ppm.

[0163] A representative ¹³C ssNMR spectrum of form J is shown in FIG.25. Form J has two chemical shift peaks at approximately 179.1±2 ppmthose being 179.6 ppm and 784.4 ppm, a set of 4 peaks at 10.0, 9.3, 8.1and 6.8 ppm and n-propanol peaks at 11.5±0.5 ppm and 25.2±0.5 ppm. Thesolvent peak can be broad and relatively weak in intensity.

[0164] A representative ¹³C ssNMR spectrum of form M is shown in FIG.26. Form M has one chemical shift peak at 179±1 ppm, being 179.6 ppm,peaks at 41.9, and 16.3 ppm, a set of 5 peaks at 10.3, 9.6, 9.3, 7.7 and7.1 ppm and an isopropanol peak at 26.0±0.5 ppm. The solvent peak can bebroad and relatively weak in intensity.

[0165] A representative ¹³C ssNMR spectrum of form N is shown in FIG.27. Form N displays chemical shifts as a combination of isomorphs inFamily I. The peaks may vary in chemical shift and in relativeintensities and width due to the mixing of variable proportion ofisomorphs contained in the form N crystalline solid solution.

[0166] A representative ¹³C ssNMR spectrum of amorphous form is shown inFIG. 28. The amorphous azithromycin displays broad chemical shifts. Thecharacteristic chemical shifts have the peak positions at 179 and 11±0.5ppm.

[0167] A summary of the observed ssNMR peaks for forms A, D, F, G, H, J,M, N and R azithromycin is given in Table 10.

Example 14 NMR Analysis of a Dosage Form

[0168] To demonstrate the ability of ¹³C ssNMR to identify the form ofazithromycin contained in a pharmaceutical dosage form, coatedazithromycin tablets containing form G azithromycin were prepared andanalyzed by ¹³C ssNMR. Tablets were wet granulated and tabletted on anF-Press (Manesty, Liverpool, UK) using 0.262″×0.531″ tooling. Tabletswere formulated and tabletted to contain 250 mg of form G azithromycinwith a total tablet weight of 450 mg using the formula given below. Thetablets were uniformly coated with pink Opadry II® (mixture of lactosemonohydrate, hydroxypropylmethylcellulose, titanium dioxide, Drug &Cosmetic red # 30, and triacetin) (Colorcon, West Point, Pa.). MaterialPercentage Batch(g) Azithromycin form “G”  58.23 174.69 Pregellatinizedcorn starch  6.00  18.00 Anhydrous dicalcium phosphate  30.85  92.55Sodium croscarmelose  2.00  6.00 Magnesium stearate with 10% sodium 2.92  8.76 laurel sulfate Total 100.00 300.00

[0169] A coated tablet was gently crushed and the powdered sample waspacked with a packing tool in solid state rotor containing no ¹³Cbackground. Analysis of the sample was performed under conditionsoutlined in Example 13.

[0170] A representative ¹³C ssNMR spectrum of the tablet containing formG azithromycin is given in FIG. 29.

Example 15 Antimicrobial Activity

[0171] The activity of the crystal forms of the present inventionagainst bacterial and protozoa pathogens is demonstrated by thecompound's ability to inhibit growth of defined strains of human (AssayI) or animal (Assays II and III) pathogens.

Assay I

[0172] Assay I, described below, employs conventional methodology andinterpretation criteria and is designed to provide direction forchemical modifications that may lead to compounds that circumventdefined mechanisms of macrolide resistance. In Assay I, a panel ofbacterial strains is assembled to include a variety of target pathogenicspecies, including representatives of macrolide resistance mechanismsthat have been characterized. Use of this panel enables the chemicalstructure/activity relationship to be determined with respect topotency, spectrum of activity, and structural elements or modificationsthat may be necessary to obviate resistance mechanisms. Bacterialpathogens that comprise the screening panel are shown in the tablebelow. In many cases, both the macrolide-susceptible parent strain andthe macrolide-resistant strain derived from it are available to providea more accurate assessment of the compound's ability to circumvent theresistance mechanism. Strains that contain the gene with the designationof ermA/ermB/ermC are resistant to macrolides, lincosamides, andstreptogramin B antibiotics due to modifications (methylation) of 23SrRNA molecules by an Erm methylase, thereby generally prevent thebinding of all three structural classes. Two types of macrolide effluxhave been described; msrA encodes a component of an efflux system instaphylococci that prevents the entry of macrolides and streptograminswhile mefAlE encodes a transmembrane protein that appears to efflux onlymacrolides. Inactivation of macrolide antibiotics can occur and can bemediated by either a phosphorylation of the 2′-hydroxyl (mph) or bycleavage of the macrocyclic lactone (esterase). The strains may becharacterized using conventional polymerase chain reaction (PCR)technology and/or by sequencing the resistance determinant. The use ofPCR technology in this application is described in J. Sutcliffe et al.,“Detection Of Erythromycin-Resistant Determinants By PCR”, AntimicrobialAgents and Chemotherapy, 40(11), 2562-2566 (1996). The assay isperformed in microtiter trays and interpreted according to PerformanceStandards for Antimicrobial Disk Susceptibility Tests—Sixth Edition:Approved Standard, published by The National Committee for ClinicalLaboratory Standards (NCCLS) guidelines; the minimum inhibitoryconcentration (MIC) is used to compare strains. The crystalline compoundis initially dissolved in dimethylsulfoxide (DMSO) as 40 mg/ml stocksolution. Strain Designation Macrolide Resistance Mechanism(s)Staphylococcus aureus 1116 susceptible parent Staphylococcus aureus 1117ErmB Staphylococcus aureus 0052 susceptible parent Staphylococcus aureus1120 ErmC Staphylococcus aureus 1032 msrA, mph, esterase Staphylococcushemolyticus 1006 msrA, mph Streptococcus pyogenes 0203 susceptibleparent Streptococcus pyogenes 1079 ErmB Streptococcus pyogenes 1062susceptible parent Streptococcus pyogenes 1061 ErmB Streptococcuspyogenes 1064 ErmB Streptococcus agalactiae 1024 susceptible parentStreptococcus agalactiae 1023 ErmB Streptococcus pneumoniae 1016Susceptible Streptococcus pneumoniae 1046 ErmB Streptococcus pneumoniae1095 ErmB Streptococcus pneumoniae 1175 MefE Streptococcus pneumoniae0085 Susceptible Haemophilus influenzae 0131 Susceptible Moraxellacatarrhalis 0040 Susceptible Moraxella catarrhalis 1055 erythromycinintermediate resistance Escherichia coli 0266 Susceptible

[0173] Assay II is utilized to test for activity against Pasteurellamultocida and Assay III is utilized to test for activity againstPasteurella haemolytica.

Assay II

[0174] This assay is based on the liquid dilution method in microliterformat. A single colony of P. multocida (strain 59A067) is inoculatedinto 5 ml of brain heart infusion (BHI) broth. The test compound isprepared by solubilizing 1 mg of the compound in 125 μl ofdimethylsulfoxide (DMSO). Dilutions of the test compound are preparedusing uninoculated BHI broth. The concentrations of the test compoundused range from 200 gg/ml to 0.098 μg/ml by two-fold serial dilutions.The P. multocida inoculated BHI is diluted with uninoculated BHI brothto make a 10⁴ cell suspension per 200 μl. The BHI cell suspensions aremixed with respective serial dilutions of the test compound, andincubated at 37° C. for 18 hours. The minimum inhibitory concentration(MIC) is equal to the concentration of the compound exhibiting 100%inhibition of growth of P. multocida as determined by comparison with anuninoculated control.

Assay III

[0175] This assay is based on the agar dilution method using a SteersReplicator. Two to five colonies isolated from an agar plate areinoculated into BHI broth and incubated overnight at 37° C. with shaking(200 rpm). The next morning, 300 μl of the fully grown P. haemolyticapreculture is inoculated into 3 ml of fresh BHI broth and is incubatedat 37° C. with shaking (200 rpm). The appropriate amounts of the testcompounds are dissolved in ethanol and a series of two-fold serialdilutions are prepared. Two ml of the respective serial dilution ismixed with 18 ml of molten BHI agar and solidified. When the inoculatedP. haemolytica culture reaches 0.5 McFarland standard density, about 5μl of the P. haemolytica culture is inoculated onto BHI agar platescontaining the various concentrations of the test compound using aSteers Replicator and incubated for 18 hours at 37° C. Initialconcentrations of the test compound range from 100-200 μg/ml. The MIC isequal to the concentration of the test compound exhibiting 100%inhibition of growth of P. haemolytica as determined by comparison withan uninoculated control.

[0176] The in vivo activity of the crystal forms of the presentinvention can be determined by conventional animal protection studieswell known to those skilled in the art, usually carried out in mice.

[0177] Mice are allotted to cages (10 per cage) upon their arrival, andallowed to acclimate for a minimum of 48 hours before being used.Animals are inoculated with 0.5 ml of a 3×10³ CFU/ml bacterialsuspension (P. multocida strain 59A006) intraperitoneally. Eachexperiment has at least 3 non-medicated control groups including oneinfected with 0.1× challenge dose and two infected with 1× challengedose; a 10× challenge data group may also be used. Generally, all micein a given study can be challenged within 30-90 minutes, especially if arepeating syringe (such as a Cornwall® syringe) is used to administerthe challenge. Thirty minutes after challenging has begun, the firstcompound treatment is given. It may be necessary for a second person tobegin compound dosing if all of the animals have not been challenged atthe end of 30 minutes. The routes of administration are subcutaneous ororal doses. Subcutaneous doses are administered into the loose skin inthe back of the neck whereas oral doses are given by means of a feedingneedle. In both cases, a volume of 0.2 ml is used per mouse. Compoundsare administered 30 minutes, 4 hours, and 24 hours after challenge. Acontrol compound of known efficacy administered by the same route isincluded in each test. Animals are observed daily, and the number ofsurvivors in each group is recorded. The P. multocida model monitoringcontinues for 96 hours (four days) post challenge.

[0178] The PD₅₀ is a calculated dose at which the compound testedprotects 50% of a group of mice from mortality due to the bacterialinfection that would be lethal in the absence of drug treatment.

[0179] The crystal forms of the present invention (hereinafter “theactive compound(s)”), may be administered through oral, parenteral,topical, or rectal routes in the treatment or prevention of bacterial orprotozoa infections. In general, the active compound is most desirablyadministered in dosages ranging from about 0.2 mg per kg body weight perday (mg/kg/day) to about 200 mg/kg/day in single or divided doses (i.e.,from 1 to 4 doses per day), although variations will necessarily occurdepending upon the species, weight and condition of the subject beingtreated and the particular route of administration chosen. However, adosage level that is in the range of about 2 mg/kg/day to about 50mg/kg/day is most desirably employed. Variations may nevertheless occurdepending upon the species of mammal, fish or bird being treated and itsindividual response to said medicament, as well as on the type ofpharmaceutical formulation chosen and the time period and interval atwhich such administration is carried out. In some instances, dosagelevels below the lower limit of the aforesaid range may be more thanadequate, while in other cases still larger doses may be employedwithout causing any harmful side effects, provided that such largerdoses are first divided into several small doses for administrationthroughout the day.

[0180] The active compound may be administered alone or in combinationwith pharmaceutically acceptable carriers or diluents by the routespreviously indicated, and such administration may be carried out insingle or multiple doses. More particularly, the active compound may beadministered in a wide variety of different dosage forms, i.e., they maybe combined with various pharmaceutically acceptable inert carriers inthe form of tablets, capsules, lozenges, troches, hard candies, powders,sprays, creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, sachets, powders for oral suspension, aqueous suspensions,injectable solutions, elixirs, syrups, and the like. Such carriersinclude solid diluents or fillers, sterile aqueous media and variousnon-toxic organic solvents, etc. Moreover, oral pharmaceuticalcompositions can be suitably sweetened and/or flavored. In general, theactive compound is present in such dosage forms at concentration levelsranging from about 1.0% to about 70% by weight.

[0181] For oral administration, tablets containing various excipientssuch as microcrystalline cellulose, sodium citrate, calcium carbonate,dicalcium phosphate and glycine may be employed along with variousdisintegrants such as starch (and preferably corn, potato or tapiocastarch), alginic acid and certain complex silicates, together withgranulation binders like polyvinylpyrrolidone, sucrose, gelatin andacacia. Additionally, lubricating agents such as magnesium stearate,sodium lauryl sulfate and talc are often very useful for tablettingpurposes. Solid compositions of a similar type may also be employed asfillers in gelatin capsules; preferred materials in this connection alsoinclude lactose or milk sugar as well as high molecular weightpolyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the active compound may be combinedwith various sweetening or flavoring agents, coloring matter or dyes,and, if so desired, emulsifying and/or suspending agents as well,together with such diluents as water, ethanol, propylene glycol,glycerin and various like combinations thereof.

[0182] For parenteral administration, solutions of the active compoundin either sesame or peanut oil or in aqueous propylene glycol may beemployed. The aqueous solutions should be suitably buffered (preferablypH greater than 8) if necessary and the liquid diluent first renderedisotonic. These aqueous solutions are suitable for intravenous injectionpurposes. The oily solutions are suitable for intraarticular,intramuscular and subcutaneous injection purposes. The preparation ofall these solutions under sterile conditions is readily accomplished bystandard pharmaceutical techniques will known to those skilled in theart.

[0183] Additionally, it is also possible to administer the activecompound topically and this may be done by way of creams, jellies, gels,pastes, patches, ointments and the like, in accordance with standardpharmaceutical practice.

[0184] For administration to animals other than humans, such as cattleor domestic animals, the active compounds may be administered in thefeed of the animals or orally as a drench composition.

[0185] The active compound may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

What is claimed is:
 1. A crystalline form of azithromycin selected fromthe group consisting of forms D, E, substantially pure F, G, J, Msubstantially in the absence of azithromycin dihydrate, N, O, P, Q, andR.
 2. A crystalline form of azithromycin according to claim 1 whereinsaid form is form D.
 3. A crystalline form according to claim 2 whereinsaid form is characterized as containing 2-6% water and 3-12%cyclohexane by weight in a powder sample.
 4. A pharmaceuticalcomposition comprising a crystalline form of azithromycin according toclaim 2 and a pharmaceutically acceptable excipient.
 5. A crystallineform of azithromycin according to claim 2 wherein said form is furthercharacterized as having a ¹³C solid state NMR spectrum comprising aplurality of peaks with chemical shifts of about 178.1 ppm, 103.9 ppm,95.1 ppm, 84.2 ppm, 10.6 ppm, 9.0 ppm and 8.6 ppm.
 6. A crystalline formof azithromycin according to claim 1 wherein said form is form E.
 7. Apharmaceutical composition comprising a crystalline form of azithromycinaccording to claim 6 and a pharmaceutically acceptable excipient.
 8. Acrystalline form of azithromycin according to claim 1 wherein said formis substantially pure form F.
 9. A crystalline form according to claim 8wherein said form is characterized as containing 2-5% water and 1-5%ethanol by weight in a powder sample.
 10. A crystalline form ofazithromycin according to claim 8 wherein said form is furthercharacterized as having a 13C solid state NMR spectrum comprising aplurality of peaks with chemical shifts of about 179.5 ppm, 178.6 ppm,58.0 ppm, 17.2 ppm, 10.1 ppm 9.8 ppm, 9.3 ppm, 7.9 ppm and 6.6 ppm. 11.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises more than 80% by weight of form F azithromycin.12. A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 81% or more by weight of form F azithromycin. 13.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 82% or more by weight of form F azithromycin. 14.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 83% or more by weight of form F azithromycin. 15.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 84% or more by weight of form F azithromycin. 16.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 85% or more by weight of form F azithromycin. 17.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 86% or more by weight of form F azithromycin. 18.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 87% or more by weight of form F azithromycin. 19.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 88% or more by weight of form F azithromycin. 20.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 89% or more by weight of form F azithromycin. 21.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 90% or more by weight of form F azithromycin. 22.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 91% or more by weight of form F azithromycin. 23.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 92% or more by weight of form F azithromycin. 24.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 93% or more by weight of form F azithromycin. 25.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 94% or more by weight of form F azithromycin. 26.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 95% or more by weight of form F azithromycin. 27.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 96% or more by weight of form F azithromycin. 28.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 97% or more by weight of form F azithromycin. 29.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 98% or more by weight of form F azithromycin. 30.A crystalline form of azithromycin according to claim 8 wherein saidazithromycin comprises 99% or more by weight of form F azithromycin. 31.A pharmaceutical composition comprising a crystalline form ofazithromycin as in claim 8 or in one of claims 11-30 and apharmaceutically acceptable excipient
 32. A crystalline form ofazithromycin according to claim 1 wherein said form is form G.
 33. Acrystalline form of azithromycin according to claim 1 wherein said formis substantially pure form G.
 34. A crystalline form according to claim32 or claim 33 wherein said form is characterized as containing 2-6%water and less than 1% organic solvent by weight in a powder sample. 35.A crystalline form of azithromycin according to claim 32 or claim 33wherein said form is further characterized as having a 13C solid stateNMR spectrum comprising a plurality of peaks with chemical shifts ofabout 179.5 ppm, 10.4 ppm, 9.9 ppm, 9.3 ppm, 7.6 ppm and 6.5 ppm.
 36. Acrystalline form of azithromycin according to claim 32 wherein saidazithromycin comprises more than 50% by weight of form G azithromycin.37. A crystalline form of azithromycin according to claim 32 whereinsaid azithromycin comprises 55% or more by weight of form Gazithromycin.
 38. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 60% or more by weight of form Gazithromycin.
 39. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 65% or more by weight of form Gazithromycin.
 40. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 70% or more by weight of form Gazithromycin.
 41. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 75% or more by weight of form Gazithromycin.
 42. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 80% or more by weight of form Gazithromycin.
 43. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 85% or more by weight of form Gazithromycin.
 44. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 90% or more by weight of form Gazithromycin.
 45. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 91% or more by weight of form Gazithromycin.
 46. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 92% or more by weight of form Gazithromycin.
 47. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 93% or more by weight of form Gazithromycin.
 48. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 94% or more by weight of form Gazithromycin.
 49. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 95% or more by weight of form Gazithr6mycin.
 50. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 96% or more by weight of form Gazithromycin.
 51. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 97% or more by weight of form Gazithromycin.
 52. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 98% or more by weight of form Gazithromycin.
 53. A crystalline form of azithromycin according to claim32 wherein said azithromycin comprises 99% or more by weight of form Gazithromycin.
 54. A pharmaceutical composition comprising a crystallineform of azithromycin according to claim 32 or claim 33 or one of claims36-53 and a pharmaceutically acceptable excipient
 55. A crystalline formaccording to claim 1 wherein said form is form H.
 56. A crystalline formof azithromycin according to claim 55 wherein said form is furthercharacterized as having a 13C solid state NMR spectrum comprising aplurality of peaks with chemical shifts of about 179.5 ppm, 178.7 ppm,9.9 ppm, 9.1 ppm, 7.9 ppm and 7.0 ppm.
 57. A pharmaceutical compositioncomprising a crystalline form of azithromycin according to claim 55 anda pharmaceutically acceptable excipient
 58. A crystalline form accordingto claim 1 wherein said form is form J.
 59. A crystalline form accordingto claim 58 wherein said form is characterized as containing 2-5% waterand 1-5% n-propanol by weight in a powder sample.
 60. A crystalline formaccording to claim 58 wherein said form is further characterized ashaving a 13C solid state NMR spectrum comprising a plurality of peakswith chemical shifts of about 179.6 ppm, 178.4 ppm, 25.2 ppm, 11.5 ppm,10.0 ppm, 9.3 ppm, 8.1 ppm and 6.8 ppm.
 61. A pharmaceutical compositioncomprising a crystalline form of azithromycin according to claim 58 anda pharmaceutically acceptable excipient.
 62. A crystalline formaccording to claim 1 wherein said form is form M substantially in theabsence of azithromycin dihydrate.
 63. A crystalline form according toclaim 62 wherein said form is characterized as containing 2-5% water and1-7% 2-propanol by weight in a powder sample.
 64. A crystalline formaccording to claim 62 wherein said form is further characterized ashaving a 13C solid state NMR spectrum comprising a plurality of peakswith chemical shifts of about 179.6 ppm, 41.9, 26.0 ppm, 16.3 ppm, 10.3ppm, 9.6 ppm, 9.3 ppm, 7.7 ppm and 7.1 ppm.
 65. A crystalline form ofazithromycin according to claim 62 wherein said azithromycin comprisesless than 5% by weight of azithromycin dihydrate.
 66. A crystalline formof azithromycin according to claim 62 wherein said azithromycincomprises less than 4% by weight of azithromycin dihydrate.
 67. Acrystalline form of azithromycin according to claim 62 wherein saidazithromycin comprises less than 3% by weight of azithromycin dihydrate.68. A crystalline form of azithromycin according to claim 62 whereinsaid azithromycin comprises less than 2% by weight of azithromycindihydrate.
 69. A crystalline form of azithromycin according to claim 62wherein said azithromycin comprises less than 1% by weight ofazithromycin dihydrate.
 70. A pharmaceutical composition comprising acrystalline form of azithromycin according to one of claims 62-69 and apharmaceutically acceptable excipient.
 71. A crystalline form accordingto claim 1 wherein said form is form N.
 72. A crystalline form accordingto claim 71 wherein said form is characterized as containing 1-5% waterand 2-5% organic solvent by weight in a powder sample.
 73. A crystallineform according to claim 71 wherein said form is further characterized ashaving a 13C solid state NMR spectrum comprising a plurality of peakswith chemical shifts of about 179.6 ppm, 178.7 ppm, 58.1 ppm, 26.0 ppm,9.9 ppm, 9.4 ppm, 7.9 ppm, and 6.6 ppm.
 74. A pharmaceutical compositioncomprising a crystalline form of azithromycin according to one of claims71-73 and a pharmaceutically acceptable excipient.
 75. A crystallineform according to claim 1 wherein said form is form
 0. 76. Apharmaceutical composition comprising a crystalline form of azithromycinaccording to claim 75 and a pharmaceutically acceptable excipient.
 77. Acrystalline form according to claim 1 wherein said form is form P.
 78. Apharmaceutical composition comprising a crystalline form of azithromycinaccording to claim 87 and a pharmaceutically acceptable excipient.
 79. Acrystalline form according to claim 1 wherein said form is form Q.
 80. Apharmaceutical composition comprising a crystalline form of azithromycinaccording to claim 79 and a pharmaceutically acceptable excipient.
 81. Acrystalline form according to claim 1 wherein said form is form R.
 82. Acrystalline form according to claim 81 wherein said form is furthercharacterized as having a 13C solid state NMR spectrum having aplurality of peaks with chemical shifts of about 177.9 ppm, 95.3 ppm,10.3 ppm, 9.6 ppm, 8.9 ppm, and 8.6 ppm.
 83. A pharmaceuticalcomposition comprising a crystalline form of azithromycin according toclaim 81 or 82 and a pharmaceutically acceptable excipient.
 84. Anazithromycin mixture comprising azithromycin dihydrate and one or morehydrate/solvates of azithromycin or azithromycin sesquhydrate.
 85. Anazithromycin mixture according to claim 84 wherein the hydrate/solvatecontains a solvent selected from the group consisting of ethanol,2-propanol, n-propanol, formic acid, butanol, pentanol, tetrahydrofuran,propylene glycol, acetone, cyclohexane, and acetonitrile
 86. Anazithromycin mixture comprising azithromycin dihydrate and one or moreforms of azithromycin selected from the group consisting of form D, formF, form G, form H, form J, form M, form 0, form Q and form R.
 87. Anazithromycin mixture according to claim 86 comprising form A and form F.88. An azithromycin mixture according to claim 86 comprising form A andform G.
 89. An azithromycin mixture according to claim 86 comprisingform A and form H.
 90. An azithromycin mixture according to claim 86comprising form A and form J.
 91. An azithromycin mixture according toclaim 86 comprising form A and form M.
 92. An azithromycin mixtureaccording to claim 86 comprising form A and form F and form G.
 93. Anazithromycin mixture according to claim 86 comprising form A and form Gand form M.
 94. A family of azithromycin crystalline forms wherein saidfamily is Family II.
 95. The family of claim 64 wherein said family ischaracterized as belonging to an orthorhombic P2₁2₁2₁ space group withcell dimensions of a=8.8±0.4 Å, b=12.3±0.5 Å and c=45±0.5 Å.
 96. Amethod of preparing the crystalline form of claim 2 comprising the stepof slurrying azithromycin with cyclohexane and isolating crystallineazithromycin.
 97. A method of preparing the crystalline form of claim 8comprising the steps of dissolving azithromycin in ethanol to form anethanol solution, cooling the ethanol solution to below 20° C.,precipitating azithromycin crystals and isolating the crystals.
 98. Amethod according to claim 97 wherein the ethanol solution is cooled to15° C. or below.
 99. A method according to claim 97 wherein the ethanolsolution is cooled to 10° C. or below.
 100. A method according to claim97 wherein the ethanol solution is cooled to 5° C. or below.
 101. Amethod as in one of claims 97-100 further comprising the step of addingwater to the ethanol solution after the ethanol solution has beencooled.
 102. A method according to claim 101 further comprising the stepof cooling the water prior to adding the water to the ethanol solution.103. A method according to claim 102 wherein the water is cooled tobelow 20° C.
 104. A method according to claim 102 wherein the water iscooled to 15° C. or below.
 105. A method according to claim 102 whereinthe water is cooled to 10° C. or below.
 106. A method according to claim102 wherein the water is cooled to 50C or below.
 107. A method accordingto one of claims 97 to 100 further comprising the step of seeding thecooled ethanol solution with crystals of form F azithromycin.
 108. Amethod of preparing the crystalline form of claim 32 comprising thesteps of dissolving azithromycin in a 1:1 mixture of water and a solventthat is a member selected from the group consisting of methanol,acetone, acetonitrile, adding water to the mixture, precipitatingazithromycin crystals and isolating the crystals.
 109. A method ofpreparing the crystalline form of claim 58 comprising the steps ofdissolving azithromycin in n-propanol, adding water, precipitatingazithromycin crystals and isolating the crystals.
 110. A method ofpreparing the crystalline form of claim 62 comprising the steps ofdissolving azithromycin with isopropanol to form an isopropanolsolution, cooling the isopropanol solution to below 15° C., adding waterafter the isopropanol solution has been cooled, precipitatingazithromycin crystals and isolating the crystals.
 111. A methodaccording to claim 110 wherein the isopropanol solution is cooled to 10°C. or below.
 112. A method according to claim 110 wherein theisopropanol solution is cooled to 5° C. or below.
 113. A methodaccording to one of claims 110 to 112 wherein the water is cooled priorto adding the water to the isopropanol solution.
 114. A method accordingto claim 113 wherein the water is cooled to 20° C. or below.
 115. Amethod according to claim 113 wherein the water is cooled to 150C orbelow.
 116. A method according to claim 113 wherein the water is cooledto 10° C. or below.
 117. A method according to claim 113 wherein thewater is cooled to 50C or below.
 118. A method according to claim 110wherein the crystals are isolated within 5 hours of precipitation. 119.A method according to claim 110 wherein the crystals are isolated within3 hours of precipitation.
 120. A method according to claim 110 whereinthe crystals are isolated within 1 hour of precipitation.
 121. A methodaccording to claim 110 wherein the crystals are isolated within 30minutes of precipitation.
 122. A method according to one of claims claim110 to 112 further comprising the step of seeding the cooled isopropanolsolution with crystals of the crystalline form of claim
 62. 123. Amethod of treating a bacterial infection or a protozoa infection in amammal, fish, or bird which comprises administering to said mammal, fishor bird a therapeutically effective amount of crystalline azithromycinaccording to claim 1 or an azithromycin mixture according to claim 86.